J. Sci. Fd Agric. 1976, 27, 733-135
Lachrymatory Factor in Sprouting Onion (Allium cepa) Chiranjib Bandyopadhyay and Gyanendra M. Tewari Biochemistry and Food Technology Division, Bhabha Atomic Research Centre, Trombay, Bombay 400 085, India (Manuscrigt received 31 December 1975)
The lachrymatory factor in dormant and sprouting onion was studied. Sprouting of onion was found to be associated with increasing yield of flavour compounds as well as lachrymators in external and internal sprouts, whereas the lachrymator content of the residual pulp remained constant during sprouting.
1. Introduction Lachrymatory factor (LF) in freshly comminuted onion has been characterised as thiopropanal S-oxidelp2 and implicated in the development of pink colour in onion homogenate.2 This factor arises as a result of rapid enzymic action of L-cysteine sulphoxide lyase (C-S-lyase) on tvuns-(+)-Spropenyl L-cysteine sulphoxide,the principal endogenous substrate known as lachrymator precursor (LP) abundantly occurring in onion bulb.3 A substantial portion of LP is present in dormant onion as a r-L-glutamyl peptide which is not acted upon by C-S-lyase and is regarded as one of the important reserve substances for nitrogen metabolism in plants.4,5 This peptide represents a major component among several other alkyl-L-cysteine sulphoxidespresent in bound form in dormant Sprouting in onion has been associated with the enhanced peptidase activity in the bulb, causing rapid hydrolysis of these peptides, whereas no such activity has been found in sprouts and in dormant onion.* Hence, in sprouting onion an accumulation of free LP derived from LP bound peptide could be expected in the bulb and such bulb on comminution should liberate more LF than dormant onion. Freeman and Whenhamg have observed an increase in flavour intensity measured in terms of thiosulphinate and other parameters in sprouted onion bulb. (Thiosulphinate was determined by measurement of U.V. absorbance of hexane extracts and has since been shown to be due to the lachrymator.10) A progressive increase in flavour intensity from outer leaf to the innermost tissue of dormant onion indicating an uneven distribution of flavour components in onion bulb has been recently reported by Freeman.11 The present work was initiated to quantify the LF of both bulb and sprouts of onion, and to demonstrate the natural distribution of LP in sprouting onion. 2. Experimental
Freshly harvested, uniformly sized red globe onions (Nasik, Maharashtra) procured from a local market, were kept for sprouting in an open plastic container at 4+ 2°C in a cool cell, illuminated with neon light. Samples of average sprout length of 3.4 cm and 22 cm were taken, after 80 and 110 days of storage respectively, segmented into three parts, i.e. internal sprout, external sprout and the remaining bulb (residual pulp). Each part was cut into small pieces, homogenised at 0°C in a Waring blendor with a small amount of water and finally extracted repeatedly with chilled peroxide-free diethyl ether. The ether extract was centrifuged to give a clear solution, washed twice with distilled water, dried over anhydrous sodium sulphate and filtered. The solvent was removed at low temperature (04°C) in a rotary evaporator and the LF content of the residue was determined as described earlier.12 Similar determination was made for unsprouted fresh onion. The lachrymator content of each extract was determined colorimetrically by measuring the extent of pink colour formation 733
734
C. Bandyopadhyay and G. M. Tewari
of the lachrymators by glycine-formaldehyde reagent according to the procedure detailed elsewhere.12 Each extract was subjected to thin-layer chromatographic (t.1.c.) separation on a silica gel G plate using light petroleum (b.p. 40-60°C): diethyl ether: acetic acid (60:40: 1, by vol.) solvent system and the lachrymator Components were identified by glycine-formaldehyde reagent .2 3. Results and discussion Table 1 summarises the yield of ether extract and lachrymator content of sprouting onion. It shows that the sprouting of onion is associated with increasing yield of ether extract of both residual pulp and sprouts, whereas the level of lachrymators in both external and internal sprouts is remarkably higher than in the residual pulp. I n contrast to the sharp changes in lachrymator content of sprouts, there is no change in that of the residual pulp during sprouting of onion. It is interesting that the lachrymator content of external sprout is appreciably higher than that of internal sprout indicating an uneven distribution of LP in sprouting onion. This observation was supported by t.1.c. separation, in which each of the samples gave identical patterns of lachrymatory components with variations of spot intensities, corresponding to lachrymator content.
Table 1. Ether extract and lachrymator content of sprouting onion kept at 4+2"Ca Lachrymator content Sample Unsprouted Sprouted
External sprout Internal sprout Residual pulp
Average Ether extract external sprout (mg/lOO g length (cm) fresh weight)
a
pmolelg fresh weight
-
90.0
8.4
0.84
3.4
203.1 161.1 105.5
18.5 10.0 8.0
4.09 1.79 0.94
208.5 223.1 163.8
21 .o 14.0 8.0
4.81 3.41 1.46
-
Sprouted
External sprout Internal sprout Residual pulp
_____
mg/100 mg ether extract
22.0
-
-
Average of three independent determinations.
Ether extract of onion represents mostly flavour compounds13 formed through C-S-lyase action on S-substituted L-cysteine sulphoxide derivatives. In sprouting onion an increased amount of extract of the residual pulp can be attributed to progressive sequential coupled reaction of peptidase and C-S-lyase on the respective substratesl4 yielding more ether soluble flavour compounds. In the sprouts, where peptidase activity is absent, the increased yield of ether extract is probably due to an increased amount of substrates for C-S-lyase activity. An increase in flavour components in onion bulb during overwinter storage has recently been demonstrated.9 I n the present study it is interesting to note the distribution of lachrymator, or in other words, of LP in three different parts of sprouting onion (Table 1). The increased yield of ether extract without any effective change in lachrymator content of residual pulp in sprouting onion apparently contradicts the well established fact of hydrolysis of LP bound peptide liberating more LP in sprouting bulb, and similarly the high content of lachrymator in sprouts, where peptidase activity is absent, is anomalous. This anomaly can perhaps be explained by suggesting that during sprouting there is a rapid translocation of excess L P (derived from LP-bound peptide) from the bulb to the sprouts possibly as a source of nitrogen in their metabolism. Thus the present report shows the possibility of utilisation of suitably sprouted onion as a richer source of flavouring than dormant bulbs.
Lachrymatory factor in sprouting onion
References 1. 2.
3. 4. 5. 6. 7. 8. 9. 10. 1 I.
12. 13. 14.
Brodnitz, M. H.; Pascale, J. V. J. agric. Fd Chem. 1971, 19, 269. Bandyopadhyay, C.; Tewari, G. M. J. agric. Fd Chem. 1973, 21, 952. Schwimmer, S. Arch. Biochem. Biophys. 1969, 130, 312. Virtanen, A. I.; Matikkala, E. J. Suomen Kemistilehti 1961, B 34, 84. Virtanen, A. I. Phytochenzistry 1965, 4, 207. Matikkala, E. J.; Virtanen, A. I. Acta Chem. Scand. 1967, 21, 2891. Virtanen, A. I. Qualities Plant. Mater Vegetabilis 1969, 18, 8. Austin, S. J.; Schwimmer, S. Enzymologia 1971,40, 273. Freeman, G. G.; Whenham, R. J. J. Sci. Fd Agric. 1974, 25, 517. Freeman, G. G.; Whenham, R. J. J. Sci. Fd Agric. 1975, 26, 1529. Freeman, G. G. J. Sci. Fd Agric. 1975, 26, 471. Tewari, G. M.; Bandyopadhyay, C. J. agric. Fd Chem. 1975, 23, 645. Bandyopadhyay, C.; Srirangarajan, A. N.; Sreenivasan, A. J. Chromatogr. 1970, 47, 400. Schwimmer, S.J. agric. Fd Chem. 1971, 19,980.
735
Lachrymatory Factor in Sprouting Onion (Allium cepa) Chiranjib Bandyopadhyay and Gyanendra M. Tewari Biochemistry and Food Technology Division, Bhabha Atomic Research Centre, Trombay, Bombay 400 085, India (Manuscrigt received 31 December 1975)
The lachrymatory factor in dormant and sprouting onion was studied. Sprouting of onion was found to be associated with increasing yield of flavour compounds as well as lachrymators in external and internal sprouts, whereas the lachrymator content of the residual pulp remained constant during sprouting.
1. Introduction Lachrymatory factor (LF) in freshly comminuted onion has been characterised as thiopropanal S-oxidelp2 and implicated in the development of pink colour in onion homogenate.2 This factor arises as a result of rapid enzymic action of L-cysteine sulphoxide lyase (C-S-lyase) on tvuns-(+)-Spropenyl L-cysteine sulphoxide,the principal endogenous substrate known as lachrymator precursor (LP) abundantly occurring in onion bulb.3 A substantial portion of LP is present in dormant onion as a r-L-glutamyl peptide which is not acted upon by C-S-lyase and is regarded as one of the important reserve substances for nitrogen metabolism in plants.4,5 This peptide represents a major component among several other alkyl-L-cysteine sulphoxidespresent in bound form in dormant Sprouting in onion has been associated with the enhanced peptidase activity in the bulb, causing rapid hydrolysis of these peptides, whereas no such activity has been found in sprouts and in dormant onion.* Hence, in sprouting onion an accumulation of free LP derived from LP bound peptide could be expected in the bulb and such bulb on comminution should liberate more LF than dormant onion. Freeman and Whenhamg have observed an increase in flavour intensity measured in terms of thiosulphinate and other parameters in sprouted onion bulb. (Thiosulphinate was determined by measurement of U.V. absorbance of hexane extracts and has since been shown to be due to the lachrymator.10) A progressive increase in flavour intensity from outer leaf to the innermost tissue of dormant onion indicating an uneven distribution of flavour components in onion bulb has been recently reported by Freeman.11 The present work was initiated to quantify the LF of both bulb and sprouts of onion, and to demonstrate the natural distribution of LP in sprouting onion. 2. Experimental
Freshly harvested, uniformly sized red globe onions (Nasik, Maharashtra) procured from a local market, were kept for sprouting in an open plastic container at 4+ 2°C in a cool cell, illuminated with neon light. Samples of average sprout length of 3.4 cm and 22 cm were taken, after 80 and 110 days of storage respectively, segmented into three parts, i.e. internal sprout, external sprout and the remaining bulb (residual pulp). Each part was cut into small pieces, homogenised at 0°C in a Waring blendor with a small amount of water and finally extracted repeatedly with chilled peroxide-free diethyl ether. The ether extract was centrifuged to give a clear solution, washed twice with distilled water, dried over anhydrous sodium sulphate and filtered. The solvent was removed at low temperature (04°C) in a rotary evaporator and the LF content of the residue was determined as described earlier.12 Similar determination was made for unsprouted fresh onion. The lachrymator content of each extract was determined colorimetrically by measuring the extent of pink colour formation 733
734
C. Bandyopadhyay and G. M. Tewari
of the lachrymators by glycine-formaldehyde reagent according to the procedure detailed elsewhere.12 Each extract was subjected to thin-layer chromatographic (t.1.c.) separation on a silica gel G plate using light petroleum (b.p. 40-60°C): diethyl ether: acetic acid (60:40: 1, by vol.) solvent system and the lachrymator Components were identified by glycine-formaldehyde reagent .2 3. Results and discussion Table 1 summarises the yield of ether extract and lachrymator content of sprouting onion. It shows that the sprouting of onion is associated with increasing yield of ether extract of both residual pulp and sprouts, whereas the level of lachrymators in both external and internal sprouts is remarkably higher than in the residual pulp. I n contrast to the sharp changes in lachrymator content of sprouts, there is no change in that of the residual pulp during sprouting of onion. It is interesting that the lachrymator content of external sprout is appreciably higher than that of internal sprout indicating an uneven distribution of LP in sprouting onion. This observation was supported by t.1.c. separation, in which each of the samples gave identical patterns of lachrymatory components with variations of spot intensities, corresponding to lachrymator content.
Table 1. Ether extract and lachrymator content of sprouting onion kept at 4+2"Ca Lachrymator content Sample Unsprouted Sprouted
External sprout Internal sprout Residual pulp
Average Ether extract external sprout (mg/lOO g length (cm) fresh weight)
a
pmolelg fresh weight
-
90.0
8.4
0.84
3.4
203.1 161.1 105.5
18.5 10.0 8.0
4.09 1.79 0.94
208.5 223.1 163.8
21 .o 14.0 8.0
4.81 3.41 1.46
-
Sprouted
External sprout Internal sprout Residual pulp
_____
mg/100 mg ether extract
22.0
-
-
Average of three independent determinations.
Ether extract of onion represents mostly flavour compounds13 formed through C-S-lyase action on S-substituted L-cysteine sulphoxide derivatives. In sprouting onion an increased amount of extract of the residual pulp can be attributed to progressive sequential coupled reaction of peptidase and C-S-lyase on the respective substratesl4 yielding more ether soluble flavour compounds. In the sprouts, where peptidase activity is absent, the increased yield of ether extract is probably due to an increased amount of substrates for C-S-lyase activity. An increase in flavour components in onion bulb during overwinter storage has recently been demonstrated.9 I n the present study it is interesting to note the distribution of lachrymator, or in other words, of LP in three different parts of sprouting onion (Table 1). The increased yield of ether extract without any effective change in lachrymator content of residual pulp in sprouting onion apparently contradicts the well established fact of hydrolysis of LP bound peptide liberating more LP in sprouting bulb, and similarly the high content of lachrymator in sprouts, where peptidase activity is absent, is anomalous. This anomaly can perhaps be explained by suggesting that during sprouting there is a rapid translocation of excess L P (derived from LP-bound peptide) from the bulb to the sprouts possibly as a source of nitrogen in their metabolism. Thus the present report shows the possibility of utilisation of suitably sprouted onion as a richer source of flavouring than dormant bulbs.
Lachrymatory factor in sprouting onion
References 1. 2.
3. 4. 5. 6. 7. 8. 9. 10. 1 I.
12. 13. 14.
Brodnitz, M. H.; Pascale, J. V. J. agric. Fd Chem. 1971, 19, 269. Bandyopadhyay, C.; Tewari, G. M. J. agric. Fd Chem. 1973, 21, 952. Schwimmer, S. Arch. Biochem. Biophys. 1969, 130, 312. Virtanen, A. I.; Matikkala, E. J. Suomen Kemistilehti 1961, B 34, 84. Virtanen, A. I. Phytochenzistry 1965, 4, 207. Matikkala, E. J.; Virtanen, A. I. Acta Chem. Scand. 1967, 21, 2891. Virtanen, A. I. Qualities Plant. Mater Vegetabilis 1969, 18, 8. Austin, S. J.; Schwimmer, S. Enzymologia 1971,40, 273. Freeman, G. G.; Whenham, R. J. J. Sci. Fd Agric. 1974, 25, 517. Freeman, G. G.; Whenham, R. J. J. Sci. Fd Agric. 1975, 26, 1529. Freeman, G. G. J. Sci. Fd Agric. 1975, 26, 471. Tewari, G. M.; Bandyopadhyay, C. J. agric. Fd Chem. 1975, 23, 645. Bandyopadhyay, C.; Srirangarajan, A. N.; Sreenivasan, A. J. Chromatogr. 1970, 47, 400. Schwimmer, S.J. agric. Fd Chem. 1971, 19,980.
735
